Named the “Method of the Year” by Nature Methods in 2010, optogenetics has been steaming forward steadily with new developments. Optogenetic tools use a combination of light and genetic engineering to control the electrical activity of a neuron with nary a patch pipette. The field has revolutionized neuroscience research by providing insights into how neurons operate singly and as members of larger networks.
Today, the nascent field of circuit optogenetics is allowing neural circuits to be studied at single-cell resolution and millisecond precision, which may further our understanding of how groups of neurons are responsible for emotions and complex behaviors. This article will look at how the development of optogenetics tools led to the birth of circuit optogenetics, and what further developments may follow.
Optogenetics in neuroscience
Optogenetics is based on the fact that a pulse of light can stimulate a type of transmembrane protein called opsin to open. In addition to being photoreceptors, opsins are also ion channels, so ions flow into the neuron when exposed to light. The class of opsins vary in their ion selectivity, wavelength of light stimulation, and ion channel kinetics. Thus one type of opsin may conduct an influx of Na+ ions upon a flash of blue light (resulting in depolarization and excitatory signals possibly sent to connected cells), while another type of opsin may conduct an influx of Cl- ions upon a flash of yellow light (resulting in hyperpolarization and inhibitory influences).
Expressing various types of opsins in neurons (by viral infection) gives scientists excitatory and inhibitory control over neurons using pulses of different wavelengths of light. Because the light pulses can be so brief, and on the scale of the neurons’ action potential firing, this system results in the ability to have unprecedented electrical control of neurons without using patch-clamping or other invasive methods. Using molecular targeting, researchers can also express the opsins of choice only in particular brain regions, or in specific types of neurons.
Circuit optogenetics
The co-evolution of opsins and optics has quickly filled the toolbox needed for the newly emerging field of circuit optogenetics, which is the study of neural circuits using optogenetics to control and study neurons selectively at single neuron resolution. The power of circuit optogenetics is readily apparent—it can assay the activity of connected neurons that are located in different brain regions. This is a way of studying brain function that is underrepresented in today’s neuroscience research methods (as opposed to macro-oriented methods like fMRI, or very focused single neuron studies). The newest tool in the optogenetics toolkit involves the light pulse used to stimulate opsins, also known as a wave front.
The complexity of designing wave fronts partly lies in the interaction between light waves and brain tissue. This challenge steepens as one tries to access neurons deeper in the brain (the main reason that initial optogenetic experiments were performed in vitro and only on surface brain layers). Wave front engineering, a field situated between biology and physics, and its application to neuroscience, is being pioneered by Valentina Emiliani, CNRS research director at the Vision Institute in Paris, head of the photonics department and the Wave Front Engineering Microscopy Group. Wave front engineering entails shaping wave fronts so that pulses of light move through brain tissue with greater precision toward their neuronal targets.
A quick flurry of advances in optical techniques recently culminated in 2-photon computer-generated holography (2-P CGH), which enables the shining of light patterns on multiple cells simultaneously. A limitation of this technique was its lack of axial resolution, however. Emiliani’s group made two further improvements that extend its utility in circuit optogenetics, using a two-step wave front coupled with temporal focusing. Temporal focusing helps to preserve the sharp borders of the stimulatory light patterns for a longer distance, up to hundreds of micrometers. This improved technique, known as multiplexed temporally focused light shaping (MTF-LS), enables scientists to stimulate neurons within the brain using a 3D pattern of light, a key ingredient for all-optical manipulation of neural circuits.
With MTF-LS, Emiliani’s group demonstrated manipulation of neural circuits in 3D for the first time. But the method could only be performed in superficial brain layers, which limits its use in studying neural circuits. They solved this problem by incorporating a GRIN lens into the MTF-LS method. A GRIN lens is a small optical probe that, when implanted into brain tissue, gives the access required to optically manipulate neurons deeper in the brain. This combination, together with today’s genetically engineered opsins, allow circuit optogenetics to study single neurons connected to one another in 3D—and deeper in the brain than ever before.
Future directions for circuit optogenetics
Emiliani’s group is currently collaborating with multiple labs to apply circuit optogenetics techniques to various model systems, including studies of short-term memory, the Zebrafish swim circuit, neurons controlling respiration, neurons in the visual system, and retinal circuits. Medical research in areas that involve neuronal circuits may soon benefit from application of circuit optogenetics, including research into Parkinson’s disease, depression, anxiety, retinal degeneration, addiction, epilepsy, memory, spinal cord injury, restoring pacemaker activity to damaged heart muscle, and controlling blood sugar levels in diabetics.
Emiliani’s lab is applying its circuit optogenetics methods to neurons of the main visual pathway. Going forward, continued progress in molecular biology, optics, biophysics, and neuroscience will fuel the emergent field of circuit optogenetics, providing previously unseen glimpses into the inner workings of circuits deep within the brain.
References
1. Multiplexed temporally focused light shaping through a gradient index lens for precise in-depth optogenetic photostimulation
2. In Vivo Submillisecond Two-Photon Optogenetics with Temporally Focused Patterned Light
3. Methods for Three-Dimensional All-Optical Manipulation of Neural Circuits